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Extraction and Analysis of Sterols in Biological Matrices by High Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry

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Extraction and Analysis of Sterols in Biological Matrices by High Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry Extraction and Analysis of Sterols in Biological Matrices by High Performance Liquid Chromatography Electrospray Ionization Mass Spectrometry Jeffrey G. McDonald, Bonne M. Thompson, Erin C. McCrum and David W. Russell. Methods in Enzymology. Volume 432, 2007, Pages 145-170 https://www.sciencedirect.com/science/article/pii/S0076687907320065 METHOD Supplies and Reagents Phosphate Buffered Saline (PBS)Cell lifters Glass culture tubes with Teflon™‐lined caps (7‐, 11‐, 14‐, 18‐ml) Wheaton Science Products 7‐ml 358640 11‐ml 358646 14‐ml 358647 18‐ml 358648 10‐ml glass pipettes Pasteur pipettes Mechanical pipette pump Glass vials with Teflon™‐lined caps (2‐, 4‐, 8‐ml) Wheaton Science Products 2‐ml W224581 4‐ml W224582 8‐ml W224584 Solid‐phase extraction (SPE) cartridges (100‐mg silica, Isolute) Vacuum apparatus for SPE 0.5% (v/v) isopropanol in hexane 30% (v/v) isopropanol in hexane HPLC‐grade methanol, ethanol, chloroform, hexane, and H2O Potassium hydroxide (10 N) Ammonium acetate Saponification solution Hyrolysis solution (6 ml of 10 N KOH diluted to 100 ml with ethanol) Bligh/Dyer solvent (1:2 [v/v] chloroform:methanol) Primary and deuterated sterol standards Luna C18 reverse‐phase HPLC column (250 × 2 mm; 3 μm‐particle size) Extraction of Lipids from Cultured Cells and Tissues Extraction of cultured cells begins by placing the culture dishes (6‐cm dishes seeded with 2e6 cells) on ice. The medium is removed with a glass pipette to a screw‐cap glass culture tube and stored for extraction later. Cells are gently washed twice with 3 ml of cold PBS, which is removed and discarded. An additional 1.6 ml of cold PBS are added to the cells, which are then gently scraped from the surface of the dish using a cell lifter. Cells are transferred to a 14‐ml screw‐cap glass culture tube. Depending on the desired information, aliquots of the medium or cells may be taken at this stage for assays to determine levels of cytokines, DNA, or protein. Lipids are extracted from the cells using a Bligh/Dyer procedure in which 6 ml of chloroform:methanol (1:2 v/v) is added to the resuspended cells in 14‐ml screw‐cap glass culture tubes. At this point, deuterated surrogate sterol standards are added for quantitative analysis, as indicated in Table 1 (see “Quantitation” section for definition of surrogate standards). Samples are vortexed at high speed for 10 s, then centrifuged at 2600 rpm (1360 rcf) for 5 min to pellet insoluble material. The supernatant is decanted to a fresh 14‐ml glass culture tubes and 2 ml each of chloroform and PBS is added. Samples are again vortexed for 10 s and centrifuged at room temperature for 5 min at 2600 rpm. Two liquid phases should now be observed. The organic lower phase is removed using a Pasteur pipette and transferred to a 4‐ml glass vial with a Teflon‐lined cap. The upper aqueous phase is discarded. We recommend the use of a mechanical pipette pump (e.g., Scienceware Pipette Pump) versus a pipette bulb for better control when removing liquid near the aqueous–organic interface. Table 1. Deuterated and primary sterols and the masses and volumes used for quantitative analysis Parts per Compounda million (PPM) Surrogate mix 1 (10 μl) 25‐Hydroxycholesterol (D3) 4 27‐Hydroxycholesterol‐(25R) (D7) 4 24,25‐Epoxycholesterol (D6) (R+S) 3 7α‐Hydroxycholesterol (D7) 4 7‐Ketocholesterol (D7) 4 4β‐Hydroxycholesterol (D7) 2 Surrogate mix 2 (10 μl) Cholesterol (D7) 150 Desmosterol (D6) 80 Internal standard (10 μl) 6α‐Hydroxycholestanol (D7) 4 Sterol mix (10 μl) 22R‐Hydroxycholesterol 4 25‐Hydroxycholesterol 4 27‐Hydroxycholesterol‐(25R) 4 24,25‐Epoxycholesterol 3 7α‐Hydroxycholesterol 4 7‐Ketocholesterol 4 4β‐Hydroxycholesterol 2 Desmosterol 80 7‐Dehydrocholesterol 100 Cholestenone 15 Cholesterol 150 Lanosterol 100 a See Table 3 for systematic sterol names and Table 4 for deuterium positions. The organic phase is evaporated under a gentle stream of dry nitrogen while heating at approximately 35° to counter the evaporative cooling effect. The dried extracts are reconstituted in approximately 200 μl of 95% (v/v) methanol, shaken gently, and transferred to an auto‐sampler vial containing the appropriate deuterated internal standard based on the amount given in Table 1 (see “Quantitation” section for definition of internal standard). An additional 200 μl of 95% methanol is added, the sample is gently shaken, and the solution is transferred to the same auto‐sampler vial. This additional rinse ensures maximum transfer of sterols from the sample vial to the auto‐sampler vial. Vortexing or sonicating the sample should not be done, as these treatments can disperse any residual insoluble material into the solution, which in turn adversely affects subsequent HPLC or MS analyses of the extracts. Occasionally, even gently shaking the extract can cause resuspension of insoluble material. If insoluble material is inadvertently resuspended, centrifugation and decanting the supernatant are usually sufficient to clarify the extract. If the final extract is not colourless and free of solid material, then the Bligh/Dyer extraction should be repeated. The auto‐sampler vial should be equipped with an appropriate volume‐reducing insert if the final volume is less than 500 μl. This extraction method is also suitable for animal tissues (brain, liver, etc.), plasma, and medium from tissue culture. Adjustments to the volumes of extraction solvents must be made to maintain the water:chloroform:methanol ratio of 1:2:0.8 for the azeotrope and a 2:2:1.8 ratio for formation of the two‐phase system. Exact volumes must be determined empirically for each sample type; however, we have found the following information useful as a guide: when extracting lipids from liquid solutions, the entire volume should be treated as the aqueous component; for tissues, the endogenous water content will contribute to the aqueous component in the azeotrope, and different tissues have different water contents. For example, an adult mouse brain (∼400 mg) will typically contribute 0.4 ml of water, meaning that 1.2 ml of PBS would be added instead of the 1.6 ml suggested above. Final volumes must also be adjusted depending on initial mass of the sample and lipid content. Tissues often require homogenization in PBS using a Potter‐Elvehjem device or a polytron prior to addition of chloroform and methanol, and again after addition of these organic solvents. The deuterated sterol standards surrogates should be added after homogenization to avoid nonspecific adsorption. Saponification of Lipid Extracts Saponifying steryl esters to free sterols requires additional steps in the extraction and purification scheme but is a more judicious use of time and resources compared to employing two HPLC‐ESI‐MS methods, which can more than double instrument time. Hydrolysis reagents should be prepared immediately prior to use according to the prescribed method already listed. The lipid extract is dried under nitrogen at approximately 35° in a glass vial with a Teflon‐lined cap, and 1 ml of hydrolysis solution is added. The vial is capped, vortexed for 10 s at maximum speed, heated to 90° for 2 h, and then allowed to cool to room temperature. Lipids are extracted from the solution using a modified Bligh/Dyer extraction in which ethanol is substituted for methanol as follows. The hydrolysed lipid solution is transferred to a 14‐ml screw‐cap glass culture tube using a Pasteur pipette, and the glass vial is rinsed once with 1 ml of ethanol. To the 2‐ml volume of hydrolysed extract in ethanol, 2 ml of chloroform and 1.8 ml of PBS are added. Samples are vortexed for 10 s at maximum speed and centrifuged at 2600 rpm for 5 min. Two phases should be observed, and the organic phase (lower) is removed using a Pasteur pipette and placed in a fresh 4‐ml glass vial with a Teflon‐lined cap, dried down, and reconstituted in 95% methanol. Solid‐Phase Extraction Bulk‐lipid extracts made with the Bligh/Dyer procedure contain many nonpolar species such as steryl esters, monoacylglycerols, diacylglycerols, and triacylglycerols, which are all strongly retained on reverse phase HPLC columns. Accumulation of these compounds on the column can lead to changes in retention time, decreased resolution, and increased back pressure. For these reasons, it is often advisable to resolve the major lipid classes by SPE prior to HPLC‐MS analysis. Sterols can be isolated from other classes of lipids by sequential development of a single SPE column and, with the appropriate solvents, oxysterols, or sterols containing additional hydroxy‐, oxo‐, or epoxy‐groups, can be separated from cholesterol and other sterols that contain a single hydroxyl functional group. For routine SPE a 100‐mg Isolute silica cartridge (Biotage, Charlottesville, VA) is prewashed by passing 2 ml of hexane through the column. A dried lipid extract prepared as previously described (with or without saponification) is dissolved in 1 ml of toluene and passed through the cartridge. Nonpolar compounds such as cholesteryl esters are eluted first with 1 ml of hexane. Cholesterol and other related sterols are eluted next with 8 ml of 30% isopropanol in hexane. The eluted sterols are dried down and then resuspended in 95% methanol prior to analysis by HPLC‐MS. To resolve sterols containing a single hydroxyl group from those containing more than one hydroxyl group (e.g., cholesterol from oxysterols), the SPE column is pre‐washed and loaded with lipids from the extraction procedure as described above. Steryl esters are again eluted from the column with 1 ml of hexane. Cholesterol and other mono‐hydroxy sterols are then eluted with 8 ml of 0.5% isopropanol in hexane. Oxysterols are eluted next with 5 ml of 30% isopropanol in hexane.
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